Cell wall core galactofuran synthesis is essential for growth of mycobacteria

The mycobacterial cell wall core consists of an outer lipid (mycolic acid) layer attached to peptidoglycan via a galactofuranosyl-containing polysacch aride, arabinogalactan. This structural arrangement strongly suggests that galactofuranosyl residues are essential for the growth and viability of myc obacteria, Galactofuranosyl residues are formed in nature by a ring contrac tion of UDP-galactopyranose to UDP-galactofuranose catalyzed by the enzyme UDP-galactopyranose mutase (Glf). In Mycobacterium tuberculosis the glf gen e overlaps, by 1 nucleotide, a gene, Rv3808c, that has been shown to encode a galactofuranosyl transferase. We demonstrate here that glf can be knocke d out in Mycobacterium smegmatis by allelic replacement only in the presenc e of two rescue plasmids carrying functional copies of glf and Rv3808c. The glf rescue plasmid was designed with a temperature-sensitive origin of rep lication and the M, smegmatis glf knockout mutant is unable to grow at the higher temperature at which the glf-containing rescue plasmid is lost. In a separate experiment, the Rv3808c rescue plasmid was designed with a temper ature-sensitive origin of replication and the glf-bearing plasmid was desig ned with a normal original of replication; this strain was also unable to g row at the nonpermissive temperature. Thus, both glf and Rv3808c are essent ial for growth. These findings and the fact that galactofuranosyl residues are not found in humans supports the development of UDP-galactopyranose mut ase and galactofuranosyl transferase as important targets for the developme nt of new antituberculosis drugs.